Despite the initial rapid progress in the discovery of the causative agent (see Chapter 2:
Virology) and the early development of diagnostic tests, further progress in the establishment of
laboratory tests for SARS has been slower than originally expected.

While various molecular (PCR-based) assays have been developed by different groups around the
world, and although one such assay is available commercially, results of these tests should still
not be used to rule out a suspected case of SARS, according to current WHO recommendations.

The continual lack of a rapid laboratory test to aid the diagnosis of suspected cases of SARS
makes this area a priority for further research efforts (WHO, Update 71).

In many viral diseases, virus shedding is greatest during the early symptomatic phase, i.e.
around, and immediately following the onset of symptoms. Unfortunately, virus excretion is
comparatively low during the initial phase of SARS. It peaks in respiratory specimens and in stools
at around day 10 after the onset of the clinical illness. In order to make an early diagnosis, it is
therefore necessary to use highly sensitive tests that are able to detect the low levels of viral
genome present during the first days of illness.

Because presently available tests are not generally able to detect the small amounts of SARS
coronavirus (SARS-CoV) initially shed, they do not yet play a role in patient management and case
control, as SARS patients may be capable of infecting others during the initial phase and therefore
need to be reliably detected and quickly isolated (WHO, Update 71).

The results of the first clinical studies on SARS are now available and able to shed light on the
clinical usefulness of various tests on different patient samples at different time points. In one
series, IgG seroconversion was documented in 93% of patients at a mean of 20 days; about 50 % of
patients had seroconverted at around 15 days after the onset of symptoms (Peiris).

In the same study, SARS-associated coronavirus RNA was detected in nasopharyngeal aspirates by
RT-PCR in 20 patients (32%) at initial presentation (mean 3.2 days after the onset of illness) and
in 68% at day 14 (Peiris).
Quantification revealed that the viral load peaked on day 10 with a mean geometric value of
1.9*107 copies per ml, compared to values of 2.3*105 copies per ml and
9.8*104 copies per ml on days 5 and 15, respectively (Peiris).

Furthermore, viral RNA was detected in 97% of stool samples collected later in the illness (a
mean of 14.2 days after onset). Similarly, viral RNA was detected in 42% of urine samples collected
at a mean of 15.2 days after the onset of symptoms (Peiris).

The authors therefore conclude that although viral RNA detection in the nasopharyngeal aspirate
has a sensitivity of only 32% at presentation, testing of multiple nasopharyngeal and fecal samples
is able to increase the predictive value of the RT-PCR assay (Peiris).

Due to the efforts of the WHO-led international multi-center collaborative network of
laboratories testing for SARS, tests for the novel coronavirus have been developed with
unprecedented speed (SARS: Laboratory diagnostic tests - 29 April 2003; http://www.who.int/csr/sars/diagnostictests/e
n/). Samples from suspected and probable SARS cases have been tested for SARS-CoV for some time
in several countries, including Canada, France, Germany, Hong Kong SAR, Italy, Japan, the
Netherlands, Singapore, the United Kingdom and the United States of America.

Nevertheless, until standardized reagents for virus and antibody detection become available and
methods have been adequately field tested, the diagnosis of SARS remains based on clinical and
epidemiological findings. The revised case definition from May 1, 2003, (see:
http://www.who.int/csr/sars/casedefinition/en/) includes laboratory results for the first time: a
suspected case of SARS, that is positive for SARS-CoV in one or more assays, should be reclassified
as a probable case. At present there are no defined criteria for SARS-CoV test results to confirm or
reject the diagnosis of SARS.

Positive laboratory test results for other known agents that are able to cause atypical pneumonia
such as Legionella pneumophila, influenza and parainfluenza viruses, Mycoplasma
pneumoniae etc. may serve as exclusion criteria: according to the case definition, a case should
be excluded if an alternative diagnosis can fully explain the illness. However, the possibility of
dual infection must not be ruled out completely.

SARS-CoV-specific RNA can be detected in various clinical specimens such as blood, stool,
respiratory secretions or body tissues by the polymerase chain reaction (PCR). A number of PCR
protocols developed by members of the WHO laboratory network are available on the WHO website
(http://www.who.int/csr/sars/primers/en/). Furthermore, a 5´-nuclease RT-PCR test kit containing
primers and positive and negative controls, developed by the Bernhard Nocht Institute (http://www.bni-hamburg.de/; Drosten et al.), is available commercially
(http://www.artus-biotech.de). An inactivated standard
preparation is also available for diagnostic purposes through the European Network for Imported
Viral Infections (ENIVD; http://www.enivd.de). ENIVD is also
preparing an international external quality assessment scheme for SARS-CoV assays.

Despite their sometimes high sensitivity, the existing PCR tests cannot rule out, with certainty,
the presence of the SARS virus in patients (Peiris, McIntosh, Poon). On the other hand, contamination of
samples in laboratories mightlead to false positive results. Stringent guidelines on
laboratory quality control and confirmatory testing have therefore been issued by the WHO (http://www.who.int/csr/sars/labmethods/en/).
P>

A valid positive PCR result indicates that there is genetic material (RNA) from the SARS-CoV in
the sample. It does not mean, however, that the virus present is infectious, or that it is present
in a large enough quantity to infect another person.

Negative PCR results do not exclude SARS. Besides the possibility of obtaining incorrect,
false-negative test results (e.g. through lack of sensitivity), specimens may not have been
collected at a time when the virus or its genetic material was present.

Currently, efforts are underway to improve the sensitivity of PCR assays to increase their
clinical usefulness. One approach is to amplify another gene of SARS-CoV than the hitherto used
polymerase gene; due to the unique transcription strategy of coronaviruses, a PCR targeting the
nucleoprotein may have a higher sensitivity. While evaluations of such a PCR are ongoing, the
protocol is already available from the Bernhard Nocht
Institute.

The presence of the infectious virus can be detected by inoculating suitable
cell cultures (e.g., Vero cells) with patient specimens (such as respiratory secretions, blood or
stool) and propagating the virus in vitro. Once isolated, the virus must be identified as SARS-CoV
using further tests. Cell culture is a very demanding test, but currently (with the exception of
animal trials) the only means to show the existence of a live virus. It has to be performed under at
least biosafety safety level (BSL) 3 conditions (see below). Positive cell culture results indicate
the presence of live SARS-CoV in the sample tested. Negative cell culture results do not exclude
SARS (see negative PCR test result).

Various methods provide a means for the detection of antibodies produced in response to
infection with SARS-CoV. Different types of antibodies (IgM and IgG) appear and change in level
during the course of infection. They can be undetectable in the early stages of infection. IgG
usually remains detectable after resolution of the illness.

The following test formats are being developed:

Enzyme-linked immunosorbent assay (ELISA): a test which detects a mixture of IgM and IgG
antibodies in the serum of SARS patients and reliably yields positive results at around day 21 after
the onset of illness.

Immunofluorescence assay (IFA): This requires the use of SARS-CoV-infected cells fixed on a
microscope slide; patient antibodies bind to viral antigens and are in turn detected by
immunofluorescent-labelled secondary antibodies against human IgG or IgM or both, using an
immunofluorescence microscope. IFA typically yields a positive result after about day 10 after the
onset of illness. Results may be quantified by using serial titrations of patient sera. A SARS-CoV
IFA manufactured by Euroimmun AG (Seekamp 31, D-23560 Lübeck, Germany; http://www.euroimmun.de) is now available commercially for the
detection of IgG and IgM antibodies against SARS-CoV.

Neutralization test (NT): This test assesses and quantifies, by means of titration, the ability
of patient sera to neutralize the infectivity of SARS-CoV on cell culture. NT is therefore likely to
be the best correlate of immunity. However, due to the use of the infectious virus it is limited to
institutions with BSL-3 facilities.

Interpretation

Positive antibody test results indicate previous infection with SARS-CoV. Seroconversion from
negative to positive or a four-fold rise in the antibody titer from acute to convalescent serum
indicates a recent infection. A negative antibody test result later than 21 days after the onset of
illness is likely to indicate that no infection with SARS-CoV has taken place. There seems to be no
background seroprevalence against SARS-CoV in the control populations screened so far. Antibody
testing allows the indirect diagnosis of SARS-CoV infection and is unsuitable during the acute
illness; it has the advantage of being rather independent of the sample type and timing, in contrast
to other virus detection methods.

All tests for SARS-CoV available so far have limitations. Extreme caution is therefore
necessary when management decisions are to be based on virological test results. For more details,
see the WHO Update 39, "Caution urged when using diagnostic tests": http://www.who.int/csr/sarsarchive/2003_04_
25/en/. In particular, false negative test results (due to low sensitivity, unsuitable sample
type, or time of sampling, etc.) may give a false sense of security; in the worst case, they could
allow persons carrying the SARS virus, and therefore capable of infecting others, to escape
detection.

To aid in the better understanding of SARS, the WHO recommends that sequential samples be stored
from patients with suspected or probable SARS - and also close contacts who are not ill themselves -
for future use. This is particularly important for the first case(s) recognized in countries that
have not previously reported SARS. Data on the clinical and contact history should also be collected
in order to obtain a better understanding of the shedding pattern of the virus and the period of
transmissibility. Such patient samples should be suitable for viral culture, PCR, antigen detection,
immunostaining and/or serological antibody assays. For details, refer to "Sampling for Severe Acute
Respiratory Syndrome (SARS) diagnostic tests", http://www.who.int/csr/sars/sampling/en/). The
WHO also encourages each country to designate a reference laboratory for investigation and/or
referral of specimens from possible SARS patients.

So far, not a single case of a laboratory-associated SARS-CoV infection has been reported.
Nevertheless, the WHO has issued biosafety guidelines for the handling of clinical specimens
associated with SARS cases and materials derived from laboratory investigations of SARS (on April
25, 2003; see http://www.who.int/csr/sars/biosafety2003
_04_25/en/). Suitable measures must be taken to prevent the potential spread by droplets, air,
and/or contaminated surfaces and objects, with particular emphasis on avoiding the unguarded
production of aerosols.

For routine diagnostic testing of serum and blood samples, manipulations involving known
inactivated (lysed, fixed or otherwise treated) virus particles and/or incomplete, non-infectious
portions of the viral genome, routine examination of mycotic and bacterial cultures, and final
packaging of specimens (already in a sealed, decontaminated primary container) for transport to
diagnostic laboratories for additional testing, BSL-2 facilities with appropriate BSL-2 work
practices are deemed sufficient. Any procedure that may generate aerosols should be performed in a
biological safety cabinet, and laboratory workers should wear eye protection and a surgical mask in
addition to standard protective equipment such as gloves, etc.

In vitro cell culture of the etiologic agent and manipulations involving growth or concentration
of the etiologic agent require BSL-3 facilities and BSL-3 work practices.

The current Dangerous Goods Regulations (2003) of the International Air Transport Association
(IATA) allow specimens known or suspected of containing the SARS agent to be transported as UN 3373
"Diagnostic Specimens" when they are transported for diagnostic or investigational purposes.
Specimens transported for any other purpose, and cultures prepared for the deliberate generation of
pathogens, must be transported as UN 2814, and marked as: "Infectious substance, affecting humans
(Severe Acute Respiratory Syndrome virus)". All specimens that are to be transported (UN 3373 or UN
2814) must be packaged in triple packaging consisting of three packaging layers.

In addition to allowing the rapid diagnosis of SARS infection, the availability of
diagnostic tests will help to address important questions such as the period of virus shedding (and
communicability) during convalescence, the presence of virus in different body fluids and excreta,
and the presence of virus shedding during the incubation period.

Until a certain degree of standardization and quality assurance has been achieved for the
SARS-CoV laboratory tests, test results must be used with utmost caution in clinical situations. It
is strongly advisable to closely check on updated recommendations by the WHO and relevant national
organizations regarding the availability and use of such tests. If in doubt, advice should be sought
from reference laboratories (see http://www.who.int/csr/sars/labmethods/en/).<
/P>